Subproject #1 Applications of 3D Whole-Cell PALM We previously developed a technique that permits super-resolution 3D imaging of whole fixed cells with genetically expressed, photoactivatable fluorescent proteins (PA-FPS, 1). Having established that the technique provides 25-50 nm lateral and 100 nm axial resolution at depths exceeding 10 microns, we are now in the process of using the microscope to assist other intramural researchers. Our collaborator Kumaran Ramamurthi (NCI) has discovered that DivIVA forms two rings at the division septum of vegetative cells, not a single ring as suggested by diffraction-limited microscopy. The spacing between the rings is 130-140 nm, just beyond the resolution of MSIM. We have successfully imaged these rings in our PALM setup, and are now investigating whether there is a double- or single ring at the forespore in sporulating cells. Our intramural (Dr. Jim Sellers, NHLBI) and extramural (Dr. Michelle Peckham, University of Leeds) collaborators have created a variety of PA-FP labeled proteins that are known to reside within the sarcomere. They are interested in using 3D PALM to visualize the interactions and relative abundance of these proteins within the Z-line. We have obtained single-color images of many of these proteins, and are now attempting to obtain dual-color super-resolution images of labeled protein pairs. Subproject #2 Selective Plane Illumination Microscopy for nematode neurodevelopment and minimally invasive imaging Selective plane illumination microscopy (SPIM (2)) is a technique whereby a sample is illuminated with a thin plane of light from the side, so that fluorescence detection occurs in a direction perpendicular to excitation. Such an experimental geometry has major advantages over conventional 3D microscopy techniques, such as confocal or 2 photon microscopy. First, acquisition speed is greatly increased relative to point-scanning methods, as the entire imaging plane is detected simultaneously. Second, excitation is confined to the focal plane, so each pixel is imaged only once during each volumetric acquisition. This drastically reduces light exposure and results in far lower photobleaching and photodamage than is possible with conventional imaging techniques. These advantages have been applied to studying whole-animal (zebrafish, drosophila) embryogenesis, and to the measurement of calcium transients in tissue slices. We have recently built a SPIM that we intend to use in constructing the first atlas of neuron positions in the developing nematode C. Elegans, in a collaboration with extramural researchers Daniel Colon-Ramos (Yale University) and Zhirong Bao (Memorial Sloan-Kettering Cancer Center). Due to the greatly reduced light dosage, we imaged nematode embryogenesis at 30x the speed of the best available competing technology (spinning disk confocal microscopy), with equivalent signal-to-noise ratio. This advance enabled the visualization of fast neurodevelopmental events in vivo (3). We have now constructed a second generation SPIM instrument, where we illuminate and detect along two perpendicular views. The advantage of this setup is that we can increase axial resolution by merging the results obtained from each view. Preliminary results suggest a tripling of axial resolution, and we are in the process of preparing a manuscript describing the application of the dual-view SPIM for improved lineaging in the nematode embryo. We are also using our existing SPIM system for collaborations with intra- and extramural collaborators. With Kanta Subbarao (NIAID), we are visualizing the movement of GFP-tagged influenza particles in living cells. With Iqbal Hamza (UMD), we are visualizing heme trafficking in living cells. For both projects, the high speed and minimal photoxicity afforded by SPIM is essential for successful imaging. Subproject 3 Multifocal Structured Illumination Microscopy Structured illumination microscopy (SIM) is a super-resolution technique (4) that offers modest resolution improvement (2x better than the diffraction limit), but is readily compatible with live samples due to its low excitation intensities. SIM, while commercially available, is expensive and remains the province of relatively few labs. Furthermore, commercial SIM systems are limited to samples with thickness < 10 microns, as they do not physically reject background light. Together with Chris Combs (NHLBI), we have developed a multifocal version of SIM that uses the confocal effect to image samples > 50 microns from the coverslip surface while maintaining resolution-doubling capability(5). The current implementation enables spatial resolutions down to 150 nm at frame rates of 1 Hz. We are working on hardware modifications to improve the speed of acquisition by 10-100 fold, and are investigating a method for further increasing spatial resolution. We have an active collaboration with Dr. George Patterson (NIBIB) to implement a multiphoton version of MSIM, thus reducing scattering and increasing penetration depth into thick samples. We are also collaborating with Dr. Clare Waterman and Dr. Robert Fischer (NHLBI) to use the current MSIM for investigating the dynamics of myosin IIA and myosin IIB in cells embedded within collagen gels. (1) York, A. et al. Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat. Methods 8, 327-333 (2011). See also code.google.com/p/palm3d/ (2) Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007-9 (2004). (3) Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 108, 17708-17713 (2011). (4) Gustafsson, M.G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 198, 82-7 (2000). (5) York, A.G. et al. Resolution Doubling in Live, Multicellular Organisms via Multifocal Structured Illumination Microscopy. Nat. Methods 9, 749-754 (2012). See also code.google.com/p/msim/